Synthesis of biotin

ABSTRACT

Synthesis of biotin from 4-carbomethoxy-2-(4,5-dihydrothiophen-3(2H)-one)-valeric acid methyl ester, and thiophene intermediates in this synthesis.

This is a division of application Ser. No. 421,460 filed Dec. 3, 1973,entitled "Synthesis of Biotin", now U.S. Pat. No. 3,978,084.

SUMMARY OF THE INVENTION

This invention is directed to a process for selectively synthesizingbiotin, which has the structural formula: ##STR1## from a4,5-dihydrothiophene compound of the formula: ##STR2## wherein R and R₁are carboxy or carboxy protected with a group convertible thereto byhydrolysis.

By means of this process, biotin can be economically produced in highyields from the 4,5-dihydrothiophene of formula I.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout this application, the term "carboxy protected with agroup convertible thereto by hydrolysis" comprehends any conventionalorganic acid protecting group which can be removed by hydrolysis. Thepreferred organic acid protecting groups are the esters. Anyconventional ester that can be hydrolyzed to yield the acid can beutilized as the protecting group. Exemplary esters useful for thispurpose are the lower alkyl esters, particularly methyl and ethylesters, the aryl esters, particularly phenyl ester, and the aryl loweralkyl esters, particularly benzyl ester.

As also used throughout this application, the term "hydrocarbyl" denotesa monovalent substituent consisting solely of carbon and hydrogen. Theterm "aliphatic" with reference to hydrocarbyl denotes straight chainand branched chain groups of 1 to 20 carbon atoms, which are saturatedor which contain one or more olefinic and/or acetylenic carbon to carbonbonds but which contain no aromatic unsaturation, such as methyl, ethyl,allyl, propargyl, hexenyl and decyl. The term "cycloaliphatic" withreference to hydrocarbyl denotes mononuclear groups of 3 to 7 carbonatoms and polynuclear groups of 7 to 17 carbon atoms, which aresaturated or which contain one or more olefinic and/or acetylenic carbonto carbon bonds but which contain no aromatic unsaturation and which cancontain one or more aliphatic hydrocarbyl moieties, such as menthyl,bornyl and cholesteryl.

As further used throughout this application, the term "lower alkyl"denotes straight chain and branched chain, saturated aliphatichydrocarbyl groups having from 1 to 8 carbon atoms, such as methyl,ethyl and propyl, preferably methyl. As also used herein, the term"aryl" signifies mononuclear aromatic hydrocarbyl groups of 6 to 13carbon atoms, such as phenyl and tolyl, which can be unsubstituted orsubstituted in one or more positions with a lower alkylenedioxy, ahalogen, a nitro, a lower alkyl or a lower alkoxy substituent, andpolynuclear aryl groups of 10 to 17 carbon atoms, such as naphthyl,anthryl, phenanthryl and azulyl, which can be substituted with one ormore of the aforementioned groups. The preferred aryl groups are thesubstituted and unsubstituted mononuclear aryl groups, particularlyphenyl. As further used herein, the term "aryl lower alkyl" comprehendsgroups wherein "aryl" and "lower alkyl" are as defined above,particularly benzyl. As still further used herein, the term "loweralkoxy" comprehends groups having from 1 to 7 carbon atoms such asmethoxy and ethoxy. Also herein, the term "halogen" or "halo", unlessotherwise stated, comprehends fluorine, chlorine, bromine and iodine.Further herein, the term "lower alkylenedioxy" comprehends groups having1 to 4 carbon atoms, such as methylenedioxy and ethylenedioxy. Stillfurther herein, the terms "loweralkylamino", "arylamino" and"arylloweralkylamino" comprehend groups wherein "aryl" and "lower alkyl"are as defined above.

As still further used throughout this application, in the pictorialrepresentations of the compounds of this application, a thickenedtapered line ( ) indicates a substituent which is in the β-orientation(above the plane of the molecule), a dotted line (---) indicates asubstituent which is in the α-orientation (below the plane of themolecule) and a wavy line ( ) indicates a substituent which is in eitherthe α- or β-orientation. It is to be undersood that the pictorialrepresentations of the compounds given throughout the specification areset forth for convenience and are to be construed as inclusive of otherforms including enantiomers and racemates and are not to be construed aslimited to the particular form shown, unless otherwise expressly stated.

In accordance with this invention, biotin is obtained by firstconverting the 4,5-dihydrothiophene of formula I to an oxime of theformula: ##STR3## wherein R and R₁ are as above. Any conventional methodof preparing an oxime from a keto compound can be used to convert the4,5-dihydrothiophene of formula I to the oxime of formula II.Preferably, the 4,5-dihydrothiophene of formula I is treated with ahydroxylamine hydrohalide, preferably hydroxylamine hydrochloride, in anitrogen-containing base. In carrying out this reaction, anyconventional nitrogen-containing base can be utilized. The preferrednitrogen-containing bases are the amines. Among the amines which can beutilized are the primary amines, such as the loweralkylamines,particularly methylamine, ethylamine, and aniline; the secondary amines,such as the diloweralkylamines, particularly dimethylamine anddiethylamine, and pyrrole; and the tertiary amines, such as thetriloweralkylamines, particularly trimethylamine and triethylamine,pyridine and picoline. Also, in carrying out this reaction with ahydroxylamine hydrohalide, temperature and pressure are not critical,and the reaction can be suitably carried out at from room temperature toreflux and at atmospheric pressure. Preferably, this reaction is carriedout at room temperature (about 22° C.). Further, this reaction can becarried out in an inert organic solvent. In this reaction anyconventional inert organic solvent can be utilized, such as thealiphatic or aromatic hydrocarbons, as for example n-hexane or benzene.Preferably, this reaction is carried out in an excess of thenitrogen-containing base, which serves as the solvent medium.

The oxime of formula II is then converted to an amine of the formula:##STR4## wherein R and R₁ are as above. This reaction is suitablycarried out by treating the oxime of formula II with a hydrohalide in aninert, organic solvent under substantially anhydrous conditions. Thisreaction can be carried out in a conventional manner, preferably bytreating the oxime of formula II with hydrogen chloride. In carrying outthis reaction, any conventional inert organic solvent can be utilized.The preferred inert organic solvents are the ethers, particularly thedilower alkyl ethers, such as diethyl ether, and the cyclic ethers, suchas tetrahydrofuran and dioxane. In carrying out this reaction,temperature and pressure are not critical, and this reaction can besuitably carried out at from 0° C. to about 70° C. and at atmosphericpressure. Preferably, this reaction is carried out at room temperature.

In carrying out the foregoing steps for converting the4,5-dihydrothiophene of formula I to the amine of formula III, it ispreferred that at least one and particularly that both of R and R₁ becarboxy protected with a group convertible thereto by hydrolysis,especially a lower alkyl ester group, particularly a methyl ester group.In accordance with this preferred aspect of the process of thisapplication, the amine of formula III, wherein R and/or R₁ are carboxyprotected with a group convertible thereto by hydrolysis, is thenconverted to an amino acid of the formula: ##STR5## In carrying out thisreaction, any conventional method of basic hydrolysis can be utilized.This hydrolysis can be suitably carried out in a conventional inertorganic solvent. The preferred solvents are the lower alkanols,particularly methanol and ethanol, and the aqueous ether solvents,preferably the aqueous dilower alkyl ethers, particularly diethyl ether,and the aqueous cyclic ethers, particularly tetrahydrofuran and dioxane.In this reaction, any conventional base can be utilized. Among thepreferred bases are the alkali metal hydroxides, such as sodium,potassium and lithium hydroxide, and the alkaline earth metalhydroxides, such as calcium and magnesium hydroxide, especially thealkali metal hydroxides. In this hydrolysis, temperature and pressureare not critical, and this reaction can be suitably carried out at fromabout 0° C. to about 100° C. and at atmospheric pressure. Preferably,this reaction is carried out at reflux, especially at about 70° C.

The amino acid of formula IV is then converted to a lactam of theformula: ##STR6## The lactam of formula V can be obtained from the aminoacid of formula IV in a conventional manner. Preferably, this reactionis carried out by heating the amino acid of formula IV in an inertorganic solvent to a temperature of from about 80° C. to about 200° C.,while removing the water formed in the reaction. In carrying out thisreaction, any conventional inert organic solvent which has a boilingpoint above about 80° C. can be utilized. Preferred inert organicsolvents include the aromatic hydrocarbons, such as benzene, xylene, andtoluene. In carrying out this reaction, particular temperatures andpressures are not critical, and this reaction can be suitably carriedout at about 100° C. and atmospheric pressure.

The lactam of formula V can also be obtained from an amine of formulaIII, wherein R₁ is carboxy and R is carboxy protected with a groupconvertible thereto by hydrolysis, i.e., a compound of the formula:##STR7## wherein R' is carboxy protected with a group convertiblethereto by hydrolysis;

by first converting the compound of formula III-A to a lactam of theformula: ##STR8## wherein R' is as above; and then hydrolyzing thelactam of formula VI. In converting the compound of formula III-A to alactam of formula VI and then hydrolyzing the lactam of formula VI toform the lactam of formula V, any conventional method for converting anamino acid to a lactam and for carrying out the basis hydrolysis of anester can be utilized. Preferably, the procedure set forth above forforming the lactam of formula V from the amino acid of formula IV andfor hydrolyzing the amine of formula III to form the amino acid offormula IV is utilized.

In accordance with this application, it is preferred to obtain thelactam of formula V from the amine of formula III by first hydrolyzingthe amine of formula III to form the amino acid of formula IV and thenconverting the amino acid of formula IV to the lactam of formula Vrather than by forming the lactam of formula VI from the compound offormula III-A and then hydrolyzing the resulting lactam of formula VI.

The lactam of formula V is then converted to mixed anhydride of theformula: ##STR9## wherein R₂ is lower alkyl or phenyl; which, in turn,is converted to an imido compound of the formula: ##STR10## wherein R₂is as above; and which, in turn, is converted to an imido-anhydride ofthe formula: ##STR11## wherein R₂ is as above. The conversion of thelactam of formula V to the compound of formula IX, via the intermediatesof formulae VII and VIII, can be carried out in a conventional manner bytreating the lactam of formula V with a lower alkyl or phenylchloroformate, preferably a lower alkyl chloroformate, in the presenceof a nitrogen-containing base. In carrying out this reaction, anyconventional nitrogen-containing base, such as the primary, secondary,and tertiary amines, set forth above, can be utilized. This reaction canbe suitably carried out in an inert organic solvent. In this reaction,any conventional inert organic solvent can be utilized, with the diloweralkyl ketones, particularly acetone, being preferred. In carrying outthis reaction, temperature and pressure are not critical, and thisreaction can be suitably carried out at from about 0° C. to about 30° C.and at atmospheric pressure. Preferably, this reaction is carried out atabout room temperature.

The imido-anhydride of formula IX is then converted to an azido-carbonylcompound of the formula: ##STR12## wherein R₂ is as above. Theimido-anhydride of formula IX can be converted to the azidocarbonylcompound of formula X by treating the compound of formula IX in aconventional manner with an alkali metal azide. This reaction can becarried out in an inert organic solvent. In this reaction, anyconventional inert organic solvent, such as the dilower alkyl ketones,can be utilized. In this reaction, temperature and pressure are notcritical, and the reaction can be suitably carried out at from about-10° C. to about +30° C. and at atmospheric pressure. Preferably, thisreaction is carried out at about 0° C.

The azidocarbonyl compound of formula X is then converted to anisocyanate of the formula: ##STR13## wherein R₂ is as above; which, inturn, is converted to a urethane of the formula: ##STR14## wherein R₂ isas above; and R₃ is aliphatic hydrocarbyl, cycloaliphatic hydrocarbyl oraryl lower alkyl;

and which, in turn, is converted to diurethane of the formula: ##STR15##wherein R₂ and R₃ are as above; and R₄ is hydroxy, lower alkoxy, aryllower alkoxy, amino, monoloweralkylamino, diloweralkylamino, arylaminoor arylloweralkylamino.

The azidocarbonyl compound of formula X can be converted to theisocyanate of formula XI and the urethane of formula XII by heating theazidocarbonyl compound of formula X in the presence of an alcohol. Inthis reaction, any primary, secondary, or tertiary alcohol can beutilized. Among the alcohols which can be utilized are the aliphatichydrocarbyl alcohols, such as ethanol, methanol, allyl alcohol,propargyl alcohol, hexenyl alcohol and decanyl alcohol, thecycloaliphatic hydrocarbyl alcohols, such as menthol, borneol andcholesterol, and the aryl lower alkanols, such as benzyl alcohol. Thisreaction can be suitably carried out in an inert organic solvent. Inthis reaction, any conventional inert organic solvent can be utilized,as for example, hexane, chloroform and benzene. Preferably, thisreaction is carried out in an excess of the alcohol, which serves as thesolvent medium. In carrying out this reaction, temperature and pressureare not critical, and temperatures of from about 50° C. to the refluxtemperature of the alcoholic mixture and atmospheric pressure can beconveniently utilized. Preferably, this reaction is carried out at fromabout 70° C. to 75° C.

In carrying out the thermolysis of the azidocarbonyl compound of formulaX to form the isocyanate of formula XI, which is in turn converted bythe alcohol to the urethane of formula XII, it is preferred to treat theazidocarbonyl compound of formula X with a lower alkanol or an aryllower alkanol. By so doing, a diurethane compound of formula XIII isobtained wherein R₃ is lower alkyl or aryl lower alkyl and R₄ is loweralkoxy or aryl lower alkoxy.

However, where the azidocarbonyl compound of formula X is treated withan alcohol other than a lower alkanol or aryl lower alkanol, it ispreferred to treat this compound with an optically active alcohol. Inthis reation step, any conventional, optically active alcohol can beutilized, such as d- or l-borneol, isopinocampheol or menthol.

Where the azidocarbonyl compound of formula X is treated with an alcoholother than a lower alkanol or aryl lower alkanol, the urethane offormula XII which results is not converted further by the alcoholutilized to the corresponding diurethane compound of formula XIII. Insuch a case, it is necessary to convert the urethane of formula XII tothe diurethane compound of formula XIII in a separate step. Theconversion of the urethane of formula XII to the diurethane compound offormula XIII, wherein R₄ is hydroxy, can be carried out by anyconventional method of hydrolyzing an imide. Preferably, the urethane offormula XII is hydrolyzed by treating it with an alkali metal hydroxideor an alkaline earth metal hydroxide, to convert it to the diurethanecompound of formula XIII. Alternatively, the urethane of formula XII canbe treated with ammonia, a mono- or di-loweralkylamine, such asmethylamine or diethylamine, an arylamine, such as phenylamine, or anarylloweralkylamine, such as benzylamine, to convert it to thediurethane compound of formula XIII, wherein R₄ is amino,monoloweralkylamino, diloweralkylamino, arylamino orarylloweralkylamino. In carrying out these reactions for obtaining thediurethane of formula XIII, temperature and pressure are not critical,and temperatures from about 50° C. to 100° C. and atmospheric pressurecan be suitably utilized. These reactions are also suitably carried outin a conventional, inert organic solvent, such as a lower alkanol,particularly methanol.

The diurethane compound of formula XIII, wherein R₄ is other thanhydroxy, whether formed by the preferred, one step treatment of theazidocarbonyl compound of formula X with a lower alkanol or aryl loweralkanol or formed by treating the urethane of formula XII with ammoniaor an amine, is then hydrolyzed with a base. This basic hydrolysis canbe carried out in a conventional manner, such as by the procedure setforth above for hydrolyzing the amine of formula III. The resultingthiophenevaleric acid has the structural formula: ##STR16## wherein R₂and R₃ are as above; and is the same valeric acid compound obtained byfirst treating the azido-carbonyl compound of formula X with an alcohol,other than a lower alkanol or aryl lower alkanol, and then hydrolyzingthe resulting urethane compound of formula XII.

The thiophenevaleric acid of formula XIII-A is then converted to atetrahydrothiophenevaleric acid of the formula: ##STR17## wherein R₂ andR₃ are as above. The tetrahydrothiophenevaleric acid formula XIV isobtained by the catalytic hydrogenation of the thiophenevaleric acidformula XIII-A in the presence of an acid. In carrying out thisreaction, any conventional, noble metal hydrogenation catalyst, such asplatinum, palladium, ruthenium or rhodium, can be utilized. Thepreferred hydrogenation catalyst is a palladium catalyst. This reactionis suitably carried out in an inert organic solvent. In this reaction,any conventional inert organic solvent in which the thiophenevalericacid compound of formula XIII-A and catalytic quantities of an acid aresoluble is suitable. Among the preferred, inert organic solvents are thelower alkanols, such as methanol and ethanol, and the cyclic ethers,such as dioxane and tetrahydrofuran. Especially preferred inert organicsolvents for carrying out this reaction are the lower alkanoic acids,particularly glacial acetic acid, in which the addition of catalyticamounts of an acid to the solvent may be dispensed with. In carrying outthis reaction in an inert organic solvent, other than an alkanoic acid,any conventional carboxylic acid may be utilized to catalyze thehydrogenation. The preferred acids for this purpose are the loweralkanoic acids, such as formic, acetic and pentanoic acid, the loweralkane dicarboxylic acids, such as succinic acid, and the aryllower-alkanoic acids, such as benzoic acid. In carrying out thisreaction, temperature and pressure are not critical, and temperaturesfrom about 25° C. to about 110° C. and pressures of from about 1,000 toabout 3,000 p.s.i. can be conveniently utilized. Preferably,temperatures of about 50° C. to 100° C., particularly about 75° C., andpressures of about 1500 to 2000 p.s.i., particularly about 1800 p.s.i.,are utilized.

In the hydrogenation of the thiophenevaleric acid of formula XIII-A inaccordance with this application, it has been found that the resultingtetrahydrothiophenevaleric acid of formula XIV is typically obtained asa racemate. However, it has been surprisingly found that thehydrogenation, as set forth above, of the compound of formula XIII-A,wherein R₃ is a residue of an optically active alcohol, formed, forexample, by heating the azidocarbonyl compound of formula X with anoptically active alcohol, yields a mixture of enantiomers of thetetrahydrothiophenevaleric acid of formula XIV in which one of theenantiomers predominates. Whether a particular R₃ enantiomer residue onthe compound of formula XIII-A will yield a tetrahydrothiophenevalericacid of formula XIV enriched in the d- or the 1-enantiomer cannot bepredicted without actually carrying out a hydrogenation with theparticular, optically active residue. However, when it is determinedthat hydrogenation of a particular compound of formula XIII-A, whichincludes a particular R₃ -enantiomer residue (e.g., a d-enantiomerresidue), yields predominately one enantiomer (e.g., an 1-enantiomer) ofthe compound of formula XIV, then it can be predicted that hydrogenationof the same compound of formula XIII-A, which includes the other R₃enantiomer residue (i.e., the 1-enantiomer residue), will yieldpredominately the other enantiomer (i.e., the d-enantiomer) of thecompound of formula XIV.

The tetrahydrothiophenevaleric acid of formula XIV, whether a racemate,an enantiomer, or a mixture of enantiomers, is then converted to biotinby the basic hydrolysis of the compound of formula XIV. The resultingbiotin will have an optical activity corresponding to the opticalactivity of the tetrahydrothiophenevaleric acid of formula XIV fromwhich it was made. This hydrolysis can be carried out in a conventionalmanner, such as by the procedure set forth above for hydrolyzing theamine of formula III. Preferably, the tetrahydrothiophenevaleric acid offormula XIV is hydrolyzed by heating it in an aqueous solutioncontaining an alkali metal hydroxide or an alkaline earth metalhydroxide. In carrying out this reaction, temperature and pressure arenot critical, and the reaction can be suitably carried out at from about50° C. to about 100° C. and at atmospheric pressure. Preferably, theaqueous solution containing the base and the tetrahydrothiophenevalericacid of formula XIV is heated to reflux.

Also, in accordance with this invention, d,1-biotin is obtained from the4,5-dihydrothiophene of formula I by first converting the lactam offormula VI to a hydrazide compound of the formula: ##STR18## The lactamof formula VI can be converted to the hydrazide of formula XV by anyconventional method of hydrazinolizing an ester. Preferably, thisreaction is carried out by treating the lactam of formula VI withhydrazine. This reaction can be carried out in an inert solvent, such aswater or a lower alkanol. Preferably, this reaction is carried out in anexcess of hydrazine, utilizing no inert solvent medium. In carrying outthis reaction, temperature and pressure are not critical, and thereaction can be suitably carried out at room temperature and atmosphericpressure.

The hydrazide compound of formula XV is then converted to anazidocarbonyl compound of the formula: ##STR19## The hydrazide offormula XV can be converted to the azidocarbonyl compound of formula XVIby a conventional, nitrous acid oxidation of carbohydrazide to anacylazide. Preferably, this reaction is carried out by treating thehydrazide of formula XV with an alkali metal nitrite in an aqueousmineral acid, especially a hydrohalic acid, particularly hydrochloricacid. In carrying out this reaction, temperature and pressure are notcritical, and this reaction can be suitably carried out at from about-20° C. to about +10° C. and at atmospheric pressure. Preferably, thisreaction is carried out at about 0° C.

The azidocarbonyl compound of formula XVI is then converted to anisocyanate of the formula: ##STR20## which is, in turn, converted to aurethane of the formula: ##STR21## wherein R₅ is aliphatic hydrocarbyl,cycloaliphatic hydrocarbyl or aryl lower alkyl.

The azidocarbonyl compound of formula XVI can be converted to theisocyanate of formula XVII, which is, in turn, converted to the urethaneof formula XVIII, by heating the azidocarbonyl compound of formula XVIin the presence of an alcohol. This reaction can be carried out in aconventional manner, such as by the procedure set forth above forconverting the azidocarbonyl compound of formula X to the urethane offormula XII. In carrying out this procedure, it is preferred to reactthe azidocarbonyl compound of formula XVI with an aliphatic hydrocarbylalcohol, a cycloaliphatic hydrocarbyl alcohol or an aryl lower alkylalcohol. Especially preferred alcohols are the lower alkyl alcohols andbenzyl alcohol.

The urethane of formula XVIII can also be obtained by first convertingthe lactam of formula V to an amide of the formula: ##STR22## andtreating and amide of formula V-A with chlorine or bromine in thepresence of an alkali metal or alkaline earth metal alkoxide orhydroxide and in the presence of an alcohol, such as an aliphatichydrocarbyl alcohol, cycloaliphatic hydrocarbyl alcohol or aryl loweralkyl alcohol.

The lactam of formula V can be converted to the amide of formula V-A byany conventional method of converting a carboxylic acid to acarboxamide. Preferably the lactam of formula V is treated first with achlorinating or brominating agent, such as thionyl chloride or thionylbromide, to form the corresponding acyl halide. This reaction can besuitably carried out in an inert organic solvent. In this reaction, anyconventional, inert organic solvent, as for example chloroform orbenzene, can be utilized. In carrying out this reaction, temperature andpressure are not critical, and this reaction can be suitably carried outat from about 25° C. to about 75° C. and at atmospheric pressure. Theresulting acyl halide is, then, preferably treated with ammonia. Thisreaction can be suitably carried out in an inert organic solvent. Inthis reaction, any conventional inert organic solvent, as for examplebenzene or chloroform, can be utilized. In this reaction, temperatureand pressure are not critical, and temperatures of about -15° C. to +50°C. and atmospheric pressure can be suitably utilized.

The amide of formula V-A is then converted to the urethane of formulaXVIII by treating it with chlorine or bromine in the presence of analkali metal or alkaline earth metal hydroxide or alkoxide and analcohol. This reaction can be suitably carried out in a conventionalmanner in an inert organic solvent. In this reaction, any conventional,inert organic solvent, as for example chloroform or benzene, can beutilized. Preferably, this reaction is carried out in an excess of thealcohol, which serves as the solvent medium. In carrying out thisreaction, temperature and pressure are not critical, and this reactioncan be suitably carried out at 25° C. to 120° C. and atmosphericpressure.

The urethane of formula XVIII is then converted to a tetrahydrothiopheneof the formula: ##STR23## wherein R₅ is as above. The urethane offormula XVIII is converted to the tetrahydrothiophene of formula XIX bythe hydrogenation in the presence of a noble metal catalyst. In carryingout this hydrogenation, the urethane is preferably hydrogenatedutilizing the procedure set forth above for hydrogenating thethiophenevaleric acid of formula XIII-A.

The tetrahydrothiophene of formula XIX is then converted to a bis-aminoacid of the formula: ##STR24## The tetrahydrothiophene of formula XIXcan be converted to the bis-amino acid of formula XX by any conventionalbasic hydrolysis whereby a lactam is converted to an amino acid. Incarrying out this basic hydrolysis, it it preferred to utilize theprocedure, set forth above, for hydrolyzing an amine of formula III.

The bis-amino compound of formula XX is then converted to d,1-biotin bytreating the bis-amino compound with phosgene. The bis-amino compound offormula XX can be treated in a conventional manner with phosgene to formd,1-biotin. Preferably, this reaction is carried out by dissolving thebis-amino compound in an aqueous base, preferably sodium carbonate, andthen introducing phosgene into the solution. In this reaction,temperature and pressure are not critical, and temperatures of fromabout -20° C. to +25° C. and atmospheric pressure can be suitablyutilized. Preferably, this reaction is carried out at about 0° C.

The biotin which is obtained by the process of this application can beobtained in pure form as the free acid, or, if desired, can beesterified in a conventional manner with a lower alkanol to form thecorresponding ester.

The 4,5-dihydrothiophene compounds of formula I, which are the startingmaterials for the process described in this application, are generallyknown. In Baker et al., J. Org. Chem., 12, 167 (1947), the preparationof 4-carbomethoxy-2-(4,5-dihydrothiophen-3(2H)-one)valeric acid methylester is described. Utilizing conventional hydrolysis andtrans-esterification procedures, this dihydrothiophene compound can beconveniently converted to form the other 4,5-dihydrothiophene compoundsof formula I of this application.

The examples which follow further illustrate this invention. Alltemperatures are in degrees Centigrade.

EXAMPLE 1

A solution of 151.4 g. (.553 mole) of4-carbomethoxy-2-(4,5-dihydrothiophen-3(2H)-one)valeric acid methylester in 470 ml. of pyridine was treated with 42.2 g. (0.608 mole) ofhydroxylamine hydrochloride, and the reaction mixture was stirred at 25°C. for 24 hours. Excess pyridine was removed on the rotary evaporator.The residue was taken up in 500 ml. of dichloromethane and washed with200 ml. of 1N hydrochloric acid. The organic layer was dried overanhydrous sodium sulfate and evaporated to dryness to yield 158.0 g.(0.546 mole, 99%) of4-carbomethoxy-2-(4,5-dihydrothiophen-3(2H)-one)valeric acid methylester oxime as a pale yellow oil.

EXAMPLE 2

Into a solution of 110 g. (0.381 mole) of4-carbomethoxy-2-(4,5-dihydrothiophen-3(2H)-one)valeric acid methylester oxime in 1500 ml. anhydrous diethyl ether, submerged in an icebath, was bubbled hydrogen chloride gas. After 3/4 hr., the flaskcontaining the reaction mixture was stoppered and the reaction allowedto proceed at 25° C. for 24 hrs. The reaction mixture was concentratedon a rotary evaporator, and the residue was taken up in 500 ml. waterand made basic by the addition of 1000 ml. 10% by weight aqueous sodiumbicarbonate solution. The reaction mixture was then extracted threetimes with 500 ml. portions of dichloromethane. The organic phases weredried over anhydrous sodium sulfate and evaporated to afford 90.0 g.(0.316 mole, 83%) of 3-amino-4-carbomethoxy-2-thiophenevaleric acidmethyl ester as a pale yellow crystalline solid; m.p. 50°-52°. Theaqueous phase was acidified with 6N hydrochloric acid to pH 4 andextracted three times with 300 ml. portions of dichloromethane. Theorganic phase was dried over anhydrous sodium sulfate and evaporated toafford 13.0 g. (0.048 mole, 13%) of3-amino-4-carbomethoxy-2-thiophenevaleric acid as a white solid m.p.130°-132°. An analytical sample was obtained by recrystallization fromethyl acetate; m.p. 131°-132°.

EXAMPLE 3

A solution of 18.64 g. (0.0687 mole) of3-amino-4-carbomethoxy-2-thiophenevaleric acid methyl ester in 400 ml.of methanol was treated with 185 ml. (0.185 mole) of 1N sodiumhydroxide. The reaction mixture was refluxed for one hour, cooled, andconcentrated. The residue, consisting essentially of3-amino-4-carbomethoxy-2-thiophenevaleric acid, was acidified to pH 1with 50 ml. 6N hydrochloric acid and evaporated to dryness leaving 16.64g. (0.0685 mole, 100%) of 3-amino-4-carboxy-2-thiophenevaleric acid as awhite solid, admixed with 7.0 g. of the sodium chloride by-product.Purification was achieved by extraction with hot ethanol. The productwas recrystallized from methanol/diethyl ether.

EXAMPLE 4

A suspension of 0.45 g. (0.00185 mole) of3-amino-4-carboxy-2-thiophenevaleric acid in 40 ml. of xylene was heatedto reflux and maintained at that point, employing a Dean-Stark trap toremove water. After one day, the system was homogeneous. The xylene wasdecanted from the polymeric by-product, which adhered to the reactionflask, and the flask was then washed with 15 ml. of hot xylene. Thexylene portions were combined and evaporated under vacuum. The residuewas partitioned between 30 ml. of dichloromethane and 60 ml. of 10% byweight aqueous sodium bicarbonate solution. The aqueous phase wasacidified to pH 1 with 6N hydrochloric acid and extracted three timeswith 30 ml. portions of dichloromethane. The organic phases werecombined, dried, and evaporated to yield 0.361 g. (0.00160 mole, 87% of3-amino-4-carboxy-2-thiophenevaleric acid-zeta-lactam as an off-whitesolid. After a recrystallization from xylene-ethanol/pet. ether, theproduct had a m.p. 215°-217° C.

EXAMPLE 5

A suspension of 15.0 g. (0.0554 mole) of3-amino-4-carbomethoxy-2-thiophenevaleric acid in 1500 ml. of xylene washeated to reflux and maintained at that temperature for one weekemploying a Dean-Stark trap to remove water. The solvent was removed onthe rotary evaporator using a high vacuum pump. The residue was taken upin 100 ml. dichloromethane and washed with 30 ml. of 10% by weightaqueous sodium bicarbonate solution. The organic layer was dried overanhydrous sodium sulfate and evaporated to afford 13.5 g. (0.0534 mole,96%) of crude 3-amino-4-carbomethoxy- 2-thiophenevaleric acid lactam.Recrystallization from ethyl acetate yielded 11.8 g. (0.0467 mole, 84%)of the product as a white solid; m.p. 167°-168° C.

EXAMPLE 6

To a solution of 4.8 g. (0.020 mole) of 3-amino-4-carbomethoxy-2-thiophenevaleric acid lactam in 60 ml. of methanol was added 28 ml.(0.022mole) of 1N sodium hydroxide. The solution was refluxed for 20minutes, cooled, and concentrated. The basic residue was extracted twicewith 75 ml. portions of methylene chloride, after the addition of 50 ml.of water. The aqueous phase was acidified to pH 1 by the addition of 25ml. of 1N hydrochloric acid and extracted three times with 75 ml.portions of dichloromethane. These dichloromethane extracts were driedover sodium sulfate, and evaporated to afford 4.3 g. (0.019 mole, 96%)of 3-amino-4-carboxy-2-thiophenevaleric acid-zeta-lactam as a whitesolid. The analytical sample, m.p. 216°-217° C., was prepared byrecrystallization from xylene-ethanol/pet. ether.

EXAMPLE 7

2.25 g. (0.010 mole) of 3-amino-4-carboxy-2-thiophenevalericacid-zeta-lactam was dissolved in 40 ml. of acetone to which 2 ml. ofwater had been added, and the solution was cooled in an ice-bath for 15minutes. At this point, 4.6 ml. (0.033 mole) of triethylamine in 40 ml.of acetone was added, followed immediately by the dropwise addition of3.3 ml. (0.033 mole) of ethyl chloroformate in 4.5 ml. of acetone over a10 minute period. The reaction mixture was stirred at 0° C. for one hourto form 3-amino-4-carbethoxyoxycarbonyl-2-thiophenevaleric acid lactam,which in turn was converted to3-carbethoxyamino-4-carboxy-2-thiophenevaleric acid lactam, which inturn was converted to3-carbethoxyamino-4-carbethoxyoxycarbonyl-2-thiophenevaleric acidlactam.

A solution of 2.13 g. (0.033 mole) sodium azide in 10 ml. of water wasthen added dropwise over a five-minute period to the reaction mixture.The reaction was further stirred at 0° C. for two hours. The reactionmixture was then partitioned between 100 ml. of dichloromethane and 75ml. of ice water. The aqueous phase was then extracted three times with30 ml. portions of dichloromethane. The organic extracts were dried overanhydrous sodium sulfate, filtered, and evaporated to leave 3.20 g.(0.009 mole, 100%) of4-azidocarbonyl-3-carbethoxyamino-2-thiophenevaleric acid lactam as acrystalline solid.

EXAMPLE 8

3.20 g. (0.099 mole) of4-azidocarbonyl-3-carbethoxyamino-2-thiophenevaleric acid lactam wasdissolved in 75 ml. of methanol, and the temperature was raised slowlyto reflux over 15 minutes. The reaction was allowed to proceed for 6hours at this temperature. Formed as intermediate in the reactionmixture was 3-carbethoxyamino-4-isocyanato-2-thiophenevaleric acidlactam, which was in turn converted to3-carbethoxyamino-4-carbomethoxyamino-2-thiophenevaleric acid lactam.The methanol was then removed, leaving 3.12 g. (0.087 mole, 87%) of3-carbethoxyamino-4-carbomethoxyamino-2-thiophenevaleric acid methylester as an oil; recrystallization from diethyl ether yielded a whitesolid; m.p. 60°-61° C.

EXAMPLE 9

0.136 g. (0.000380 mole) of3-carbethoxyamino-4-carbomethoxyamino-2-thiophenevaleric acid methylester was dissolved in 5 ml. of methanol and treated with 0.55 ml. of 1Nsodium hydroxide. The reaction mixture was refluxed for three hours, andthen cooled, and the methanol was then evaporated. The residue was takenup in 30 ml. of dichloromethane and treated with 30 ml. of water. Afterseparating the process, the aqueous phase was acidified with 2 ml. of 1Nhydrochloric acid and extracted three times with 30 ml. portions ofdichloromethane. The organic phases were cooled, dried over anhydroussodium sulfate, and evaporated to yield 0.129 g. (0.000376 mole, 99%) of3-carbethoxyamino-4-carbomethoxyamino-2-thiophenevaleric acid as a whitesolid. An analytical sample was prepared by recrystallization frommethanol; m.p. 159°-160° C.

EXAMPLE 10

0.344 g. (0.001 mole) of3-carbethoxyamino-4-carbomethoxyamino-2-thiophenevaleric acid wasdissolved in 200 ml. of glacial acetic acid and subjected to 1800 psi.hydrogen gas in a steel autoclave at 50° C. for 10 hrs., in the presenceof 2.0 g. of 10% Pd/C catalyst. After cooling to room temperature (22°C.), the autoclave was vented, and the catalyst was filtered and washedwith 100 ml. of glacial acetic acid. The solvent was removed undervacuum to afford 0.328 g. (0.00095 mole, 95%) of all cisd,1-3-carbethoxyamino-4-carbomethoxyamino-2-tetrahydrothiophenevalericacid as a colorless oil.

EXAMPLE 11

0.208 g. (0.0006 mole) of all cisd,1-3-carbethoxyamino-4-carbomethoxyamino- 2-tetrahydrothiophenevalericacid was dissolved in 1.8 ml. (0.0018 mole) of 1N sodium hydroxide. Thereaction solution was refluxed for 4.0 hrs. At this point, the pH wasadjusted to 1 by the addition of 3 ml. of 1N hydrochloric acid. Thesolvent was removed to leave a tan crystalline residue. Upon addition of5 ml. of water, a solid remained out of solution, which was filtered anddried to yield 0.050 g. (0.00021 mole, 35%) of d,1-biotin, which couldbe recrystallized from water; m.p. 232°-233° C.

EXAMPLE 12

15.0 g. (0.0628 mole) of 3-amino-4-carbomethoxy-2-thiophenevaleric acidlactam was dissolved at 25° C. in 25 ml. of 95% hydrazine. After 10minutes, the product began to crystallize. The reaction was allowed toproceed for 1/2 hr., and then, the reaction mixture was concentrated.The residue was filtered and washed with cold ethanol to afford 15.0 g.(0.0628 mole, 100%) of 3-amino-4-carbazoyl-2-thiophenevaleric acidlactam; m.p. 191°-192° C.

An analytical sample was prepared by recrystallization from ethanol.

EXAMPLE 13

To a solution of 12.24 g. (0.052 mole) of3-amino-4-carbazoyl-2-thiophenevaleric acid lactam in 200 ml. of 1Nhydrochloric acid was added dropwise at 0° C. 4.4 g. (0.062 mole) ofsodium nitrite in 30 ml. of water (previously cooled to 0°) over a14-minute period. The heterogeneous mixture was stirred for 1/2 hr., andthe mixture was further extracted five times with 50 ml. portions ofchloroform. The extracts were dried over anhydrous sodium sulfate andevaporated to afford 13.0 g. (0.052 mole, 100%) of3-amino-4-azidocarbonyl-2-thiophenevaleric acid lactam as a colorlessoil.

EXAMPLE 14

13.0 g. (0.052 mole) of 3-amino-4-azidocarbonyl-2-thiophenevaleric acidlactam was dissolved in 500 ml. of dry methanol, and the temperature wasraised to 50° C. After 15 min. at that temperature, the solution wascarefully brought up to reflux. The rate of heating was determined bythe amount of vigorous gas evolution. The solution was refluxed for 6.0hrs. Formed as an intermediate in the reaction mixture was3-amino-4-isocyanato-2-thiophenevaleric acid lactam. The solution wasthen cooled and evaporated to afford 12.5 g. (0.0491 mole, 95%) of3-amino-4-carbomethoxyamino-2-thiophenevaleric acid lactam as a whitecrystalline solid; m.p. 208°-210° C.

EXAMPLE 15

1.0 g. (0.004 mole) of 3-amino-4-carbomethoxyamino-2-thiophenevalericacid lactam was dissolved in 200 ml. of glacial acetic acid and placedin a steel autoclave. After addition of 2.0 g. of 10% Pd/C catalyst thereaction mixture was hydrogenated at 100° C. and 1800 psi for 10.0 hrs.The autoclave was cooled, vented, and the catalyst was filtered andwashed with 100 ml. of acetic acid. The solvent was removed, and the cisd,1-3-amino-4-carbomethoxyamino-2-tetrahydrothiophenevaleric acid lactamresidue was taken up in 50 ml. of water to which 10.0 g. Ba(OH)₂.sup..8H₂ O had been added.

The reaction mixture was refluxed for 20.0 hours and cooled. Carbondioxide was bubbled in until the pH dropped to 4. The precipitatedbarium carbonate was filtered and washed with 20 ml. of water. Thefiltrate was acidified with 1N sulfuric acid until acid turned to congored. The precipitated barium sulfate was filtered. The filtrate wasevaporated to dryness, and the residue was taken up in 120 ml. of 10% byweight sodium carbonate and cooled to 0° C. Gaseous phosgene was thenbubbled in for 25 minutes until the medium was acidic to congo red.After 2.0 hrs., an impurity was filtered, and the filtrate wasevaporated to dryness. The d,1-biotin residue was suspended in 70 ml. ofdry methanol and treated with 1 drop of sulfuric acid. The mixture wasrefluxed to one hour, cooled, and filtered. The salt was washed threetimes with 10 ml. portions of methanol. The filtrate was evaporated, andthe residue was partitioned between 50 ml. of chloroform and 20 ml. ofwater. The aqueous phase was extracted three times with 20 ml. portionsof chloroform. The organic phases were combined, dried over anhydroussodium sulfate, and evaporated to give 0.300 g. (.00116 mole, 29%) ofcrude d,1-biotin methyl ester. The mixture was taken up in 3 ml. ofdichloromethane and plated on three, thick layer silica plates. Elutionwas with 10% by volume methanol/chloroform solution. After two elutions0.042 g. (0.000163 mole, 4%) of pure d,1-biotin methyl ester, m.p.131°-132° C., was obtained by removal of the band at R_(f) =0.26.

EXAMPLE 16

A suspension of 2.25 g. (0.010 mole) of3-amino-4-carboxy-2-thiophenevaleric acid-zeta-lactam in 200 ml. ofchloroform was treated with 10 ml. of thionyl chloride. The reaction wasrefluxed for 2.0 hrs., cooled and evaporated to afford3-amino-4-chlorocarbonyl-2-thiophenevaleric acid lactam as a yellowsolid. The yield was quantitative after drying under high vacuum.

EXAMPLE 17

Ammonia was bubbled into a solution of 2.60 g (0.010 mole) of 3-amino-4-chlorocarbonyl-2-thiophenevaleric acid lactam in 200 ml of benzene at0° C. for 20 minutes. The solution was then stirred at 25° C. for 2.0hrs. and evaporated. The solid residue was suspended in water andfiltered to afford pure 3-amino-4-carboxamido-2-thiophenevaleric lactamas a cream-colored solid quantitatively yield.

EXAMPLE 18

3.56 g (0.010 mole) of4-azidocarbonyl-3-carbethoxyamino-2-thiophenevaleric acid lactam wasdissolved in 100 ml benzene. To this solution was added 3.08 g (0.020mole) of (+)-isopinocampheol, and the reaction was brought to reflux andmaintained at that temperature for 16 hrs. After cooling, the reactionwas concentrated to afford 7.0 g of a residue, containing4-pinyloxyamino-3-carbethoxyamino- 2-thiophenevaleric acid lactam whichwas up in 175 ml of methanol. The solution was treated with 50 ml of 1Nsodium hydroxide and subsequently refluxed 0.5 hr. After cooling to 25°C., a solid impurity was filtered off, and the filtrate wasconcentrated. The residue was partitioned between 30 ml of diethyl etherand 50 ml of 1N sodium hydroxide. The ether phase was concentrated toafford 1.8 g of recovered alcohol. The aqueous phase was acifified to pH1 with 1N HCl and extracted three times with 30 ml portions ofdichloromethane. The organic extracts were dried over sodium sulfate andevaporated to yield 3.55 g (0.0076 mole, 76 %) of 3-carbethoxyamino-4-carbo-3-pinyloxyamino-2-thiophenevaleric acid as a viscous oil.

EXAMPLE 19

0.466 g (0.001 mole) 3-carbethoxyamino- 4-carbo-3-pinyloxyamino-2-thiophenevaleric acid was hydrogenated in 200 ml of acetic acid at 50°C/1800 psi over a 10 hr. period. The catalyst loading was 2.0 g. Theautoclave was cooled overnight, and the catalyst was filtered. Thefiltrate was evaporated to dryness to afford 0.460 g (0.001 mole, 100%)of all-cis-3-carbethoxyamino-4-carbo-3-pinyloxyamino-2-tetrahydrothiophenevaleric acid as a colorlessoil.

EXAMPLE 20

0.455 g (0.96 mmole) of the all-cis-3-carbethoxy-4carbo-3-pinyloxyamino-2-tetrahydrothiophenevaleric acid, prepared in Example 18, was dissolvedin 3.5 ml of 1N sodium hydroxide. After dilution with 3 ml. of water,the reaction was refluxed for 5.0 hrs., cooled, and partitioned between20 ml ethylacetate and 10 ml water. After three 10 ml extractions withethyl acetate, the aqueous phase was acifified with 1N HCl to pH l andconcentrated. Upon cooling, the concentrate deposited 0.080 g (0.328mole, 34%) of biotin. The compound was collected by filtration andrecrystallized from water. [α] _(D) ²⁵ =+ 4°, corresponding to anoptical induction of 4.5% with an enantiomeric excess of d-biotin.

EXAMPLE 21

3.56 g (0.010 mole) of 4-azidocarbonyl-3-carbethoxyamino-2-thiophenevaleric acid lactam was dissolved in 100 ml of benzene. Tothis solution was added 3.08 g (0.020 mole) of l-borenol, and thereaction was then refluxed overnight, cooled, concentrated, and taken upin 250 ml of methanol. The solution containing3-carbethoxyamino-4-carbo-2-1-bornyloxyamino-2-thiophenevaleric acidlactam, was treated with 50 ml of 1N sodium hydroxide and refluxed 0.45hr. The reaction was cooled and concentrated, and the residue waspartitioned between 30 ml of diethyl ether and 50 ml of 1N sodiumhydroxide. The ether phase was discarded and the aqueous phase wasacidified to pH 1 with 1N HCl. The mixture was extracted three timeswith 20 ml portions of dichloromethane. The organic extracts were driedover sodium sulfate and evaporated by yield 2.76 g (0.0059 mole, 59%) of3-carbethoxyamino-4-carbo-2-1-bornyloxy-amino-2-thiophenevaleric acid asa colorless foam.

EXAMPLE 22

0.466 g (.001 mole) of 3-carbethoxyamino-4-carbo-2-1-bornyloxyamino-2-thiophenevaleric acid was hydrogenated in 200 ml of acetic acid at 50°C/1800 psi over a 10 hr. period. The catalyst loading was 2.0 g. Theautoclave was cooled overnight, and the catalyst was filtered. Thefiltrate was evaporated to dryness to yield 0.0470 g (100%) ofall-cis-4-carbo-2-bornyloxyamino-3-carbethoxyamino-2-tetrahydrothiophenevaleric acid asa colorless oil.

EXAMPLE 23

0.0470 g (0.001 mole) of the all cis-4-carbo-2-1-bornyloxyamino- 3-carbethoxyamino-2-tetrahydrothiophenevaleric acid prepared in Example22, was dissolved in 4 ml of 1N sodium hydroxide. After dilution with 5ml of water, the reaction was allowed to reflux overnight. The reactionmixture was cooled, extracted with ethyl acetate, and the aqueous phasewas acidified to pH 1 with 1N HCl. After a further extraction with ethylacetate, the aqueous phase was evaporated to dryness. The residue wasdissolved in 25 ml of 10% by wt. potassium carbonate, and phosgene wasbubbled in until the pH was acid to congo red. The mixture was againevaporated to dryness. The residue was suspended in 50 ml of methanoland two drops of conc. sulfuric acid were added. After a reflux periodof 1 hour, the reaction was cooled and evaporated. The residue waspartitioned between 30 ml of methylene chloride and 20 ml of water. Theaqueous layer was further extracted three times with 25 ml portions ofmethylene chloride. The organic phases were combined, dried over sodiumsulfate, and evaporated to yield .085 g of crude d,1-biotin methylester. Purification was achieved by chromatography on silica; thesolvent system was benzene/ethyl acetate/acetic acid (60:35:5 parts byvolume). From the chromatogram, 0.040 g (0.085 mmole, 8.5%) ofcrystalline biotin methyl ester was isolated. [α] _(D) ²⁵ =-5° (c =0.40, CH₃ OH), corresponding to an optical induction of 7% with anenantiomeric excess of 1-biotin.

EXAMPLE 24

0.0356 g (0.001 mole) of 4-aziodcarbonyl-3-carbethoxyamino-2-thiophenevaleric acid lactam was dissolved in 20 mlof benzene. After the addition of 0.344 g (0.0022 mole) of 1-menthol,the reaction mixture was refluxed 1.5 hrs., cooled, and evaporated. The3-carbethoxyamino-4 -carbo-p-menth-3-yloxyamino-2 -thiophenevaleric acidlactam residue was taken up in 20 ml of methanol and treated with 5 mlof 1N sodium hydroxide. After a reflux period of 2.5 hrs., the reactionwas cooled, concentrated, and the residue was partitioned between 20 mlof diethyl ether and 40 ml of 1N sodium hydroxide. The ether phase,containing excess 1-menthol, was discarded, and the aqueous layer wasacidified to pH 1 with 1N HCl. The mixture was then extracted threetimes with 20 ml portions of dichloromethane. The organic extracts weredried over sodium sulfate and evaporated to afford a residue, which waschromatographed on thick layer silica plates (solvent system: 5 % byvolume CH₃ OH/CHCl.sub. 3). From the chromatogram, 0.400 g (0.0086 mole,86%) of3-carbethoxyamino-4-carbo-p-menth-3-yloxy-amino-2-thiophenevaleric acidwas isolated as a colorless viscous oil.

EXAMPLE 25

0.200 g (0.427 mmole) of3-carbethoxyamino-4-carbo-p-menth-3-yloxyamino-2-thiophenevaleric acid 1was hydrogenated in 200 ml of acetic acid at 50° C/1800 psi over a 10hr. period. The catalyst loading was 2.0 g 10% Pd/C. The autoclave wascooled overnight, and the catalyst was filtered. The filtrate wasevaporated to dryness to leave 0.200 g (0.427 mmole, 100%) of allcis-3-carbethoxyamino-4-carbo-p-menth-3-yloxyamino-2-tetrahydrothiophenevaleric acid 2, as a colorless oil.

EXAMPLE 26

0.200 g (.45 mmole) of the all cis-3-carbethoxyamino-4-carbo-p-menth-3-yloxyamino-2-tetrahydrothiophenevaleric acid, prepared in Example 25,was dissolved in a mixture of 1.5 ml of 1N sodium hydroxide and 0.0 mlof water. The system was refluxed for 5.0 hrs. Upon cooling, somementhol was deposited. The reaction mixture was partitioned between 10ml of 1N sodium hydroxide and 20 ml of methylene chloride. After anadditional two 10 ml extractions with dichloromethane, the aqueous layerwas acidified with 1N HCl to pH 1. After filtration of an insolubleimpurity, the filtrate was evaporated to dryness. The residue wassuspended in 50 ml of dry methanol, treated with 2 drops of conc.sulfuric acid, and refluxed 0.45 hr. The mixture was then concentrated,and the residue was partitioned between 20 ml of methylene chloride and50 ml of water. The aqueous layer was further extracted three times with20 ml portions of methylene chloride. The organic phases were combineddried over sodium sulfate, and evaporated to yield 0.030 g (0.123 mmole,27%) of biotin methyl ester. After recrystallization from methanoldiethyl ether, a specimen of m.p. 128°-129° C. was obtained. [α]_(D) ²⁵=-3.8°, which corresponds to an optical induction of 5%, with anenantiomeric excess of 1-biotin.

We claim:
 1. A compound of the formula: ##STR25## wherein R₅ is astraight or branched chain aliphatic hydrocarbyl group having from 1-20carbon atoms; a cycloaliphatic group selected from the group consistingof menthyl, bornyl and cholesteryl and aryl lower alkyl groups whereinthe aryl moiety is selected from the group consisting of phenyl, tolyl,naphthyl, phenanthryl and azulyl wherein said aryl moieties may beunsubstituted or substituted with lower alkylenedioxy, halogen, loweralkyl, lower alkoxy or a nitro group and the lower alkyl moiety is astraight or branched chain alkyl group having 1 to 8 carbon atoms. 2.The compound of claim 1 wherein said compound is allcis-d,1-3-amino-4-carbomethoxyamino-2-tetrahydrothiophenevaleric acidlactam.